Many species of bacteria control gene expression on a community-wide scale by producing, secreting, detecting and responding to extracellular signaling molecules (sometimes called ‘autoinducers’ or ‘pheromones’) that accumulate in the environment. This phenomenon is termed ‘quorum sensing’ (QS) as gene expression is triggered by the ‘sensing’ of the pheromone when its concentration has reached a ‘quorum’. In Gram-positive bacteria, the signaling molecules are mainly short peptides acting either from the outside part of bacteria or from the inside, after internalization via oligopeptide transport systems called Opp or Ami.
Several bacterial functions such as the virulence in Staphylococcus aureus and Enterococcus faecalis, the competence in Bacillus subtilis or the production of bacteriocin in Lactococcus lactis are controlled by peptides acting at the surface of bacteria. However, the paradigm for mechanisms involving peptides detected from the outside is the regulation of the competence state for natural transformation by chromosomal donor DNA in Streptococcus pneumoniae. In this bacterium, the signaling peptide, called CSP (Competence Stimulating Peptide) and encoded by the gene comC, is secreted and matured by an ABC transporter, ComAB. The detection of the extracellular CSP at the surface of the bacterium is achieved by a two component system (TCS). Firstly, the membrane embedded histidine kinase, ComD, autophosphorylates in response to CSP and further phosphorylates its cognate response regulator, ComE, which activates transcription of a few genes, the early CSP-induced genes, including comCDE, comAB and comX. ComX is an alternative competence specific sigma factor required for expression of late CSP-induced genes, which comprise genes encoding the DNA uptake machinery.
Concerning signaling peptides that are active after internalization by an oligopeptide transporter, three groups have been described in detail: (i) Phr peptides in B. subtilis involved in the control of sporulation, competence, and production of degradative enzymes and antibiotics, (ii) PapR peptides involved in the control of virulence of bacteria belonging to the Bacillus cereus group and (iii) peptides involved in the control of plasmid transfer in Enterococcus faecalis. All these extracellular short peptides interact with either Rap phosphatases (in B. subtilis) or transcriptional regulators (PlcR in B. cereus or PrgX in E. faecalis) to elicit a physiological response.
Oligopeptide transport systems involved in these signaling pathways belong to the superfamily of ATP-binding cassette (ABC) transporters. They are composed of five subunits: an extracellular oligopeptide-binding protein, OppA that specifically captures the substrates, two transmembrane proteins, OppB and OppC that form the pore and two membrane-bound cytoplasmic ATP-binding proteins, OppD and OppF that provide the energy for peptide translocation. Several copies of the opp operon and/or of the genes encoding the oligopeptide-binding proteins can be present in a single genome. The genome of Streptococcus thermophilus encodes one oligopeptide transport system and depending on the strain, two (strain LMD-9 and CNRZ1066) or three (strain LMG18311) oligopeptide-binding proteins. In Gram-positive bacteria, two main functions have been attributed to the Opp transporters: nutrition and sensing. The nutritional role has been well studied in lactic acid bacteria such as Lactococcus lactis or S. thermophilus. During growth in milk, the Opp transporters supply these auxotrophic bacteria with peptides that serve as amino acid sources. The sensing function is more complex and is poorly documented, particularly in nonpathogenic bacteria.
Among the nonpathogenic bacteria, S. thermophilus is of major importance for the food industry since it is massively used for the manufacture of yoghurt and Swiss or Italian-type cheeses with an annual market value of approximately $40 billion making S. thermophilus a species of major economic importance. The industry is continuously working to improve the properties of S. thermophilus starter strains. Even though the fermentation properties of this bacterium have been gradually improved by classical methods, there is great potential for further improvement through genetic engineering.
However, until now, only genetic tools based on genetically modified bacteria exist. For example, Havarstein has disclosed an inducible system (stb system) that permits the surexpression of proteins in S. thermophilus (Blomgvist T. et al “Pheromone-induced expression of recombinant proteins in Streptococcus thermophilus” Arch Microbiol. 2006 December; 186(6):465-73. Epub 2006 Aug. 24.). In particular, this article discloses that a possible peptide-pheromone (STP) regulates bacteriocin production in S. thermophilus LMG 18311, and shows that the StbABCHR (system that regulates bacteriocin production) quorum-sensing system can be exploited for inducible expression of recombinant proteins in this bacterial species.
Thus, there is a need for an efficient method that allows obtaining improved Firmicutes, in particular Streptococcus, and more preferably S. thermophilus by natural processes of gene transfer, and not artificial gene transfer. This is particularly important for the food industries, for example the dairy industries which do not want to use GMO in their products.
Regarding this need of genetic tools, competence is poorly understood in S. thermophilus. In fact, regarding QS systems, only one of them has been yet described, which QS system (called stb or blp) controls the production of a bacteriocin.
The sequencing of the genome of three strains of S. thermophilus, CNRZ1066, LMG18311 and LMD-9, has revealed the presence of comX and of 14 proteins with strong similarities with the 14 proteins known to be required for competence in S. pneumoniae and encoded by late CSP-induced genes (Bolotin et al., Complete sequence and comparative genome analysis of the dairy bacterium Streptococcus thermophilus., Nat. Biotechnol. 22:1554-1558, 2004; Makarova et al. Comparative genomics of the lactic acid bacteria. Proc Natl Acad Sci USA., 103(42):15611-6, 2006). Except comX, no ortholog of the early CSP-induced genes of S. pneumoniae have been detected in the genome of S. thermophilus. It has been shown that overexpression of comX induces the competent state in S. thermophilus LMG18311.
Still, how transformation is turned on in this strain and what regulatory pathway and more especially which competence stimulating peptide (CSP) controls the expression of comX have not previously been explained.